U.S. patent application number 12/467755 was filed with the patent office on 2010-01-28 for shielded cross-linking probes.
Invention is credited to Niles A. Pierce, Jeff Vieregg, Peng Yin.
Application Number | 20100021904 12/467755 |
Document ID | / |
Family ID | 41568981 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100021904 |
Kind Code |
A1 |
Pierce; Niles A. ; et
al. |
January 28, 2010 |
SHIELDED CROSS-LINKING PROBES
Abstract
The present invention relates to the use of nucleic acid probes
to bind to targets. In some embodiments, the probe comprises a
shielded cross-linking probe.
Inventors: |
Pierce; Niles A.; (Pasadena,
CA) ; Yin; Peng; (Pasadena, CA) ; Vieregg;
Jeff; (Studio City, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
41568981 |
Appl. No.: |
12/467755 |
Filed: |
May 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61128411 |
May 21, 2008 |
|
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|
Current U.S.
Class: |
435/6.11 ;
435/91.1; 436/94; 536/24.3; 536/25.3 |
Current CPC
Class: |
C12Q 1/6832 20130101;
C12Q 1/6832 20130101; C12Q 2525/161 20130101; C12Q 2525/186
20130101; Y10T 436/143333 20150115; C12Q 2523/101 20130101 |
Class at
Publication: |
435/6 ; 536/24.3;
536/25.3; 436/94; 435/91.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C07H 1/00 20060101
C07H001/00; G01N 33/00 20060101 G01N033/00; C12P 19/34 20060101
C12P019/34 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0003] This invention was made with government support under grant
nos. NIH 5R01 EB006192-04 "Hybridization chain reaction: in situ
amplification for biological imaging" and NIH P50 HG004071 "Center
for in toto genomic analysis of vertebrate development".
Claims
1. A cross-linking probe comprising: an initiator region; a probe
region, wherein the probe region is linked to the initiator region;
at least one cross-linker that is part of the probe region; and a
blocking region that is hybridized to the probe region such that
the blocking region reduces the cross-linking of the cross-linker
to other molecules when the blocking region is hybridized to the
probe region.
2. The cross-linking probe of claim 1, further comprising a loop
region that links the probe region to the blocking region.
3. The cross-linking probe of claim 1, wherein the cross-linker is
an activatable cross-linker.
4. The cross-linking probe of claim 1, wherein the activatable
cross-linker is light activatable.
5. The cross-linking probe of claim 1, wherein the activatable
cross-linker is conformationally activatable.
6. The cross-linking probe of claim 1, wherein the probe region
comprises a first subprobe region and a second subprobe region.
7. The cross-linking probe of claim 1, wherein the probe region
comprises at least on nucleotide on both sides of the
cross-linker.
8. The cross-linking probe of claim 1, further comprising a
detectable marker.
9. The cross-linking probe of claim 8, wherein the detectable
marker is attached to a detectable marker region.
10. The cross-linking probe of claim 1, further comprising a
detectable marker, wherein the detectable marker is linked to an
amplifier molecule, and wherein the amplifier molecule is
cross-linked to the cross-linking probe.
11. The cross-linking probe of claim 1, wherein the cross-linking
probe further comprises a pairing region.
12. The cross-linking probe of claim 11, wherein the cross-linking
probe is cross-linked to an amplifier molecule, wherein the
amplifier molecule comprises: a complementary pairing region that
selectively hybridizes to at least a part of the pairing region,
wherein the complementary pairing region comprises a cross-linker;
a blocking region that can hybridize to the complementary pairing
region and can dissociate from the complementary pairing region;
and a detectable marker region linked to the complementary pairing
region, wherein the detectable marker region comprises one or more
detectable markers.
13. The cross-linking probe of claim 1, wherein the probe further
comprises a pairing region.
14. The cross-linking probe of claim 13, where the cross-linking
probe is cross-linked to an amplifier molecule, wherein the
amplifier molecule comprises: a complementary pairing region that
selectively hybridizes to the pairing region; and a detectable
marker region, comprising one or more detectable markers.
15. The cross-linking probe of claim 13, wherein the pairing region
comprises one or more orthogonal nucleotides.
16. The cross-linking probe of claim 13, wherein there are no
consecutive natural bases in the pairing region.
17. The cross-linking probe of claim 1, wherein the cross-linker
comprises an extender linker.
18. The cross-linking probe of claim 1, wherein the probe region is
immediately adjacent to the initiator region.
19. The cross-linking probe of claim 1, further comprising
detectable marker region that comprises at least one fluorescent
label.
20. The cross-linking probe of claim 1, further comprising a first
monomer that is cross-linked to the probe region.
21. The cross-linking probe of claim 20, further comprising a
second monomer crosslinked to the first monomer.
22. A method of associating a cross-linking probe with a nucleic
acid sequence, said method comprising: providing a cross-linking
probe and a nucleic acid sequence; wherein the cross-linking probe
comprises: an initiator region; a probe region, wherein the probe
region is linked to the initiator region; at least one cross-linker
that is part of the probe region; and a blocking region hybridized
to the probe region; hybridizing the initiator region to a part of
the nucleic acid sequence; dissociating the blocking region from
the probe region; hybridizing the probe region to a second part of
the nucleic acid sequence; and cross-linking the cross-linker.
23. The method of claim 22, further comprising performing an
ultrastringent wash following cross-linking.
24. The method of claim 22, wherein the cross-linking probe further
comprises a detectable marker.
25. The method of claim 24, further comprising the step of
detecting the presence or absence of the detectable marker
following the ultrastringent wash.
26. The method of claim 25, wherein the detectable marker is
attached to a detectable marker region.
27. The method of claim 26, wherein the detectable marker region is
linked to the blocking region.
28. The method of claim 22, wherein the cross-linking probe is
associated with a detectable marker region, wherein the detectable
marker region is part of an amplifier molecule, and wherein the
amplifier molecule is cross-linked to the cross-linking probe.
29. The method of claim 22, wherein the cross-linking probe is
associated with a detectable marker region, wherein the detectable
marker region is part of an amplifier molecule, and wherein the
amplifier molecule is hybridized to the cross-linking probe,
wherein the amplifier molecule comprises one or more orthogonal
nucleotides.
30. The method of claim 22, wherein the method is performed in a
cell.
31. The method of claim 22, wherein the method is performed in
vitro.
32. The method of claim 22, wherein the method is performed in
vivo.
33. A method of determining the presence or absence of a nucleic
acid, said method comprising: providing a sample; adding to the
sample an initiator region that is linked to a probe region,
wherein there is at least one uncross-linked cross-linker that is
part of the probe region, and wherein a blocking region is
hybridized to the probe region when the initiator region is added
to the sample; hybridizing the initiator region to a first part of
a nucleic acid contained within the sample, if the nucleic acid is
present in the sample; dissociating the blocking region from the
probe region, if the nucleic acid is present in the sample;
hybridizing the probe region to a second part of the nucleic acid,
if the nucleic acid is present in the sample; cross-linking the
cross-linker; performing a wash following the cross-linking;
associating the probe region with a detectable marker; and
detecting the presence or absence of the detectable marker, thereby
determining the presence or absence of a nucleic acid.
34. The method of claim 33, wherein the detectable marker is linked
to a detectable marker region that is linked to the blocking
region.
35. The method of claim 33, wherein the probe region is linked to a
pairing region, and wherein the detectable marker is attached to a
first monomer that is crosslinked to the pairing region.
36. The method of claim 35, further comprising a second monomer
that is crosslinked to the first monomer.
37. The method of claim 33, wherein the detectable marker is
attached to an amplifier molecule that comprises a pairing region
that hybridizes to a complementary pairing region that is linked to
the initiator region, wherein the pairing region comprises at least
a second cross-linker.
38. The method of claim 37, wherein the amplifier molecule is added
after the wash, and wherein following the addition of the amplifier
molecule, the second cross-linker is cross-linked and a second wash
is performed.
39. The method of claim 37, wherein the detectable marker is
attached to an amplifier molecule that comprises a pairing region
that hybridizes to a complementary pairing region that is linked to
the initiator region, wherein the pairing region comprises at least
one orthogonal nucleotide.
40. The method of claim 37, wherein the initiator region and the
probe region are no longer than 50 nucleotides in length.
41. The method of claim 37, wherein the cross-linking probe is no
longer than 1000 nucleotides in length.
42. The cross-linking probe of claim 4, wherein the activatable
cross-linker comprises psoralen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application No. 61/128,411, filed May 21, 2008, which is hereby
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled Seq_Listing_CALTE-049A, created May 18, 2009, which
is 688 bytes in size. The information in the electronic format of
the Sequence Listing is incorporated herein by reference in its
entirety.
BACKGROUND
[0004] 1. Field
[0005] The present disclosure relates to various probes that can
bind and cross-link to a target nucleic acid.
[0006] 2. Background
[0007] Molecules that selectively bind to nucleic acids have a
large variety of uses, including modifying gene regulation and/or
protein expression, as well as being able to serve as indicators
for the presence or absence of a specific nucleic acid in a sample.
One example of an indicator role is in the technique of in situ
hybridization, which allows for the spatial mapping of nucleic acid
sequences, such as mRNAs, in normal and pathological tissues. In
situ hybridization can be used to study gene expression and
regulation in a morphological context from the sub-cellular to the
organismal levels (Lawrence, J. B., R. H. Singer, and L. M.
Marselle, Highly localized tracks of specific transcripts within
interphase nuclei visualized by in situ hybridization. Cell, 1989.
57: p. 493-502; Kislauskis, E. H., et al., Isoform-specific
3'-unstranslated sequences sort alpha-cardiac and beta-cytoplasmic
actin messenger RNAs to different cytoplasmic compartments. The
Journal of Cell Biology, 1993. 123(1): p. 165-172; Wilkie, G. S.,
et al., Transcribed genes are localized according to chromosomal
position within polarized Drosophila embryonic nuclei. Current
Biology, 1999. 9: p. 1263-1266; Levsky, J. M., et al., Single-cell
gene expression profiling. Science, 2002. 297: p. 836-840; Qian, X.
and R. V. Lloyd, Recent developments in signal amplification
methods for in situ hybridization. Diagnostic Molecular Pathology,
2003. 12(1): p. 1-13; Qian, X., L. Jin, and R. V. Lloyd, In situ
hybridization: basic approaches and recent development. The Journal
of Histotechnology, 2004. 27(1): p. 53-67; Kosman, D., et al.,
Multiplex detection of RNA expression in Drosophila embryos.
Science, 2004. 305: p. 846.)
SUMMARY OF THE INVENTION
[0008] In some aspects, probes and/or methods involving a molecular
conformational change is/are used to provide an enhanced level of
specificity for the formation of cross-links between a probe and
its target. In some embodiments, these cross-links then allow for
the use of ultra-stringent washing to eliminate the remaining
probes from the sample.
[0009] In some aspect, a new approach to in situ hybridization is
provided. It achieves high specificity using methods that, in some
embodiments, are suitable for sensitive, multiplexed, quantitative
bioimaging in fixed cells, tissue sections, and whole-mount
embryos.
[0010] In some aspects, probes and/or methods involving the
selective displacement of a blocking region from a probe that
contains a cross-linker are provided. The displacement of the
blocking region can allow for superior selectivity in creating a
cross-link between a probe and a target nucleic acid.
[0011] In some aspects, a cross-linking probe is provided that
comprises an initiator region; a probe region, wherein the probe
region is linked to the initiator region; at least one cross-linker
that is part of the probe region; and a blocking region that is
hybridized to the probe region such that the blocking region
reduces the cross-linking of the cross-linker to other molecules
when the blocking region is hybridized to the probe region.
[0012] In some aspects, a method of associating a cross-linking
probe with a nucleic acid sequence is provided that comprises
providing a cross-linking probe and a nucleic acid sequence;
wherein the cross-linking probe comprises: an initiator region; a
probe region, wherein the probe region is linked to the initiator
region; at least one cross-linker that is part of the probe region;
and a blocking region hybridized to the probe region. The method
can further comprise hybridizing the initiator region to a part of
the nucleic acid sequence; dissociating the blocking region from
the probe region; hybridizing the probe region to a second part of
the nucleic acid sequence; and cross-linking the cross-linker.
[0013] In some aspects, a method of determining the presence or
absence of a nucleic acid is provided and comprises providing a
sample; adding to the sample an initiator region that is linked to
a probe region, wherein there is at least one uncross-linked
cross-linker that is part of the probe region, and wherein a
blocking region is hybridized to the probe region when the
initiator region is added. The method can further comprise
hybridizing the initiator region to a first part of a nucleic acid
contained within the sample, if the nucleic acid is present in the
sample; dissociating the blocking region from the probe region, if
the nucleic acid is present in the sample; hybridizing the probe
region to a second part of the nucleic acid, if the nucleic acid is
present in the sample; cross-linking the cross-linker; performing a
wash following the cross-linking; associating the probe region with
a detectable marker; and detecting the presence or absence of the
detectable marker, thereby determining the presence or absence of a
nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A depicts some embodiments of a cross-linking
probe.
[0015] FIG. 1B is a flow chart outlining some embodiments of using
a cross-linking probe.
[0016] FIG. 2 depicts some embodiments of using a cross-linking
probe.
[0017] FIG. 3 depicts an embodiment of making a cross-linking
probe.
[0018] FIG. 4 depicts some embodiments of a cross-linking
probe.
[0019] FIG. 5 depicts some embodiments of a cross-linking
probe.
[0020] FIG. 6 depicts an embodiment of an HCR method employing a
cross-linking probe.
[0021] FIG. 7 depicts two different photoactivatable crosslinkers
and the gel results from Example 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0022] The present disclosure provides a wide variety of
alternative cross-linking probes and various methods of using them.
In some embodiments, these probes and techniques provide the
ability to create covalent bonds to their target nucleic acid and
to do so with a high degree of selectivity and/or specificity. In
some embodiments, these probes and techniques allow for the
selective cross-linking of the probe to a specific nucleic acid. In
some embodiments, the cross-linking itself is selective in that
cross-linking between unbound cross-linking probes and non-target
(and non-probe) nucleic acids is significantly reduced or
prevented.
[0023] In some embodiments, a shielded cross-linking probe includes
a probe region, a cross-linker (that is in or part of the probe
region) and a blocking region that reduces the ability of the
cross-linker to cross-link and/or the probe region to bind to
undesired sequences. The blocking region will dissociate from the
probe region in the presence of the target nucleic acid, which then
allows the cross-linker to cross-link to the desired target. In
some embodiments, the selectivity of the cross-linker is enhanced
further by the use of an activatable cross-linker, which allows one
to determine when and/or where in a reaction, sample, or system the
cross-linker should become capable of cross-linking. The above
selective cross-linking can provide a variety of useful functions.
For example, the specifically cross-linked probe can be used in an
in situ hybridization technique which can then employ
ultrastringent washing conditions in order to obtain superior in
situ results. Of course, any process in which very specific and
very tight (e.g., covalent level strength) binding between a
molecule and a target nucleic acid are beneficial can benefit from
one or more of the embodiments disclosed herein.
[0024] In some embodiments, shielded cross-linking probes will form
a crosslink to a target (such as mRNA) with high specificity by
employing one or more of the following: 1) initial sequestration of
one or more activatable cross-linkers within a duplex portion of a
nucleic acid probe; 2) stringent sequence filtering via competitive
branch migration replacement of blocker/probe region base pairs
with probe/target base pairs such that upon completion of the
triggered conformation change, the cross-linkers are sequestered
within a new probe/target duplex; and 3) high-yield activation of
the cross-linkers. These properties, especially when more than one
is present, will ensure that the probe covalently cross-links to
the sample primarily (and in some embodiments only) when it is
specifically base-paired to its complementary target.
[0025] The following section outlines the definitions of some of
the terms used herein as well as providing some alternative
embodiments. Following that section, a general description of how
the cross-linking probes can be used and the various components is
provided. Following that section, a description of various
alternative embodiments, which also includes a description of
variations of the various elements disclosed herein, is provided.
Following that section, a series of Examples outlining some
possible uses of some of the disclosed embodiments is provided.
DEFINITIONS
[0026] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including but not limited to, patents,
patent applications, articles, books, treatises, and internet web
pages are expressly incorporated by reference in their entirety for
any purpose. When definitions of terms in incorporated references
appear to differ from the definitions provided in the present
teachings, the definition provided in the present teachings shall
control. It will be appreciated that there is an implied "about"
prior to the temperatures, concentrations, times, etc discussed in
the present teachings, such that slight and insubstantial
deviations are within the scope of the present teachings herein. In
this application, the use of the singular includes the plural
unless specifically stated otherwise. Also, the use of "comprise",
"comprises", "comprising", "contain", "contains", "containing",
"include", "includes", and "including" are not intended to be
limiting. It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention.
[0027] Unless otherwise defined, scientific and technical terms
used in connection with the invention described herein shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Further, unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular. Generally, nomenclatures utilized in
connection with, and techniques of, cell and tissue culture,
molecular biology, and protein and oligo- or polynucleotide
chemistry and hybridization described herein are those well known
and commonly used in the art. Standard techniques are used, for
example, for nucleic acid purification and preparation, chemical
analysis, recombinant nucleic acid, and oligonucleotide synthesis.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The techniques and
procedures described herein are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the instant specification. See, e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual
[0028] (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2000). The nomenclatures utilized in connection with,
and the laboratory procedures and techniques of described herein
are those well known and commonly used in the art.
[0029] As utilized in accordance with the embodiments provided
herein, the following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0030] The term "nucleic acid" refers to natural nucleic acids,
artificial nucleic acids, non-natural nucleic acid, orthogonal
nucleotides, analogs thereof, or combinations thereof. Nucleic
acids may also include analogs of DNA or RNA having modifications
to either the bases or the backbone. For example, nucleic acid, as
used herein, includes the use of peptide nucleic acids (PNA). The
term "nucleic acids" also includes chimeric molecules.
[0031] As used herein, the terms "polynucleotide,"
"oligonucleotide," and "nucleic acid oligomers" are used
interchangeably and mean single-stranded and double-stranded
polymers of nucleic acids, including, but not limited to,
2'-deoxyribonucleotides (nucleic acid) and ribonucleotides (RNA)
linked by internucleotide phosphodiester bond linkages, e.g. 3'-5'
and 2'-5', inverted linkages, e.g. 3'-3' and 5'-5', branched
structures, or analog nucleic acids. Polynucleotides have
associated counter ions, such as H.sup.+, NH.sub.4.sup.+,
trialkylammonium, Mg.sup.2+, Na.sup.+ and the like. A
polynucleotide can be composed entirely of deoxyribonucleotides,
entirely of ribonucleotides, or chimeric mixtures thereof.
Polynucleotides can be comprised of nucleobase and sugar analogs.
Polynucleotides typically range in size from a few monomeric units,
e.g. 5-40 when they are more commonly frequently referred to in the
art as oligonucleotides, to several thousands of monomeric
nucleotide units. Unless denoted otherwise, whenever a
polynucleotide sequence is represented, it will be understood that
the nucleotides are in 5' to 3' order from left to right and that
"A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine.
[0032] The term "hairpin" refers to a structure formed by
intramolecular base pairing in a single-stranded polynucleotide
ending in an unpaired loop. A "hairpin loop" refers to a single
stranded region that loops back on itself and is closed by a single
base pair.
[0033] The term "initiator region" denotes a section of a probe
that can initially bind to a target nucleic acid, or at least a
part of the target nucleic acid. In some embodiments, the initiator
region is generally two to 1000 nucleotides, such as 3-100
nucleotides or 4-50 nucleotides. In some embodiments, there can be
more than one initiator. For example, in some embodiments there can
be two initiators, one on each side of the probe region.
[0034] The term "probe region" denotes a section of a hybridization
probe that binds to a target nucleic acid. In some embodiments,
this is distinguishable from the initiator region in that an
initiator region, if present, will bind to the target first. This
initial binding allows for the initiation of the displacement of
the blocking region via a branch migration process. In some
embodiments the probe region is 3-1000 nucleotides in length, such
as 6-100 or 10-50 nucleotides in length.
[0035] The term "linked" or "links" denotes that two regions are
covalently connected to one another. There can be additional
intervening structures between the two regions. Thus, the linking
can be direct (also described as being "immediately adjacent" ) or
indirect. While two nucleic acids that become cross-linked could be
characterized as "linked," for the sake of clarity, such links are
referred to herein as "cross-links" and not generally denoted by
the term "link". Thus, a single nucleic acid that includes a probe
region, a cross-linker, a loop region, and a blocking region would
all be "linked" to each other, and if the cross-linker cross-linked
to the blocking region, then the cross-linker (and the probe region
that the cross-linker is in) would be "cross-linked" to the
blocking region.
[0036] The term "associated" denotes that the relevant structures
and/or regions are localized with one another by some type of
binding interaction. Association can be due to covalent bonds
(e.g., linked or cross-linked) or they can be due to noncovalent
bonds (e.g., hybridization, antibody binding, etc.)
[0037] The term "cross-linked" denotes that a cross-linker has
formed a covalent bond with another residue, molecule, nucleotide,
etc. The bond can be formed within the same molecule (e.g.,
cross-linking a hairpin loop shut) or can be between molecules
(e.g., cross-linking a cross-linking probe to a target molecule).
"Cross-linked" can include one or more cross-linked bonds.
[0038] The term "cross-linker" denotes a molecule or atom that is
capable of forming a covalent bond to another molecule. While the
cross-linkers should allow for two separate molecules (e.g.,
nucleic acids) to be effectively cross-linked together, more than
one cross-linker can be used, thus, any single cross-linker does
not need to be strong enough to keep the two molecules bonded
together. In some embodiments, the cross-linker is capable of
forming a covalent bond between the target nucleic acid and a probe
region.
[0039] The term "blocking region" denotes a structure that
obstructs the probe region and cross-linker when it is hybridized
to the probe region. While the blocking region need not completely
prevent any interaction of the cross-linker and/or probe region
with the environment, it will significantly reduce this interaction
so as to allow a greater degree of specificity of cross-linking
that will depend upon the presence or absence of the blocking
region. The blocking region is displaced via a branch migration in
which base-pairs between the probe region and blocking region are
replaced one at a time by base pairs between the probe region and
the target. Hence, the cross-linkers are shielded (and in some
embodiments are always effectively shielded) from the biological
sample either within the probe or between the probe and the
intended target. In some embodiments, the blocking region contains
one or more areas that are inert to cross-linking. For example, the
blocking region can contain a subregion that is just the polymer
backbone without any bases. The blocking region can be a single
strand of nucleotides or one or more strands of nucleotides.
[0040] The term "reversibly hybridize" denotes that the molecule
can hybridize and dissociate (or be displaced from) from a nucleic
acid. Nucleic acids that are cross-linked to one another will not
dissociate due to the covalent interaction. However, molecules that
include cross-linkers that have not been activated can still
reversibly hybridize or dissociate from a complementary molecule.
In addition, a molecule can be described as being "reversibly
hybridizable," even if it has a cross-linker in it, as long as it
can dissociate from a binding molecule.
[0041] The term "reduce" denotes some decrease in amount. In some
embodiments, an event is reduced by 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,
99, 99.9, 99.99, 99.999, percent or more, including any value above
any of the preceding values, as well as any range defined between
any two of the preceding values.
[0042] The term "loop region" denotes a region that link a probe
region and a blocking region. This region is optional. The loop
region can include nucleotides and/or simply be a polymer backbone
without any bases.
[0043] The modifier "sub" denotes a component of a region.
[0044] The term "detectable marker" denotes any molecules that can
be observed and/or detected, either directly (e.g., fluorescence),
or indirectly (e.g., detection of a product, where the presence of
the product is based upon the presence of a target). For indirect
detectable markers, the molecule that acts as the intermediate can
be the "detectable marker," even if it is a product from that
molecule that is actually detected. In some embodiments, a
detectable marker is a part of the probe that can serve as a
marker, such as in a surface plasmon resonance technique (such as
in a BIACORE.TM. machine). Thus, in some embodiments, no separate
detectable marker need be included.
[0045] The term "amplifier molecule" denotes a molecule that allows
for the association of a detectable marker with a probe region.
This association can be indirect, e.g., through an additional
pairing region. The amplifier can be cross-linked to the
cross-linking probe, but need not be.
[0046] The term "pairing region" denotes a nucleic acid sequence
that can hybridize to a complementary pairing region.
[0047] The term "complementary pairing region" denotes a nucleic
acid sequence that can hybridize to a pairing region.
[0048] The term "orthogonal nucleotides" denotes those nucleotides
that will not effectively basepair with a natural nucleotide.
Natural nucleotides are defined as adenine, cytosine, guanine,
thymine, and uracil. Some types of orthogonal nucleotides contain
modified bases, such as isoC and isoG.
[0049] The term "external linker" denotes one or more atoms that
serve to link two molecules together. A cross-linker can be
attached to or include an external linker, which can allow for
greater flexibility in the positioning of the cross-linker. The
term "external" is used to further distinguish this linking aspect
from a cross-linker (which is capable of forming a cross-link), as
an external linker need not be capable of actually forming a
cross-link after it has been connected and can merely serve as a
longer tether to attach, for example, the cross-linker to the probe
region.
[0050] The term "activated" denotes that the cross-linker has been
exposed to a stimulus that promotes cross-linking.
[0051] The term "activatable" denotes that a cross-linker has a
state dependent ability to cross-link. In some embodiments, the
"state" is an environmental factor, such as radiation (UV, visible,
etc.), presence of a particular chemical compound, or a
conformational change (such as the presence of absence of the
blocker region).
[0052] The term "unactivated" denotes that an activatable
cross-linker has not been activated, and thus, the likelihood that
a cross-link will be formed is very low.
[0053] The term "un-cross-linked" denotes that the cross-linker has
not been cross-linked. A molecule can be activated and still be
uncross-linked.
[0054] The term "ultrastringent wash" denotes a washing condition
that is much more effective at weakening hybridization interactions
than those commonly used for in situ hybridization. Examples
include elevated temperatures and high concentrations of chemical
denaturants such as formamide, organic cosolvents, or urea. In some
embodiments, an ultrastringent wash would completely eliminate
nucleic acid hybridization in the sample, thus causing any
non-crosslinked probes to dissociate from their binding sites. In
some embodiments, an ultrastingent wash would typically be one that
is strong enough to destabilize all base-pairing (including
orthogonal base pairs) between the probe and the sample. This could
be achieved using higher temperatures or (e.g. high concentration
of formamide). In some embodiments, an ultrastringent wash
comprises 2.times.SSC and 75% formamide at 70 degrees. In some
embodiments, 20-50% formamide is employed instead of 75%. In some
embodiments, these wash conditions are for relatively short probes
of less than 1000 nucleotides, for example, 900, 800, 700, 600,
500, 400, 300, 200, 100, 50, 40, 30, 20, 10 or fewer nucleotides,
including any range defined between any two of the preceding
values.
[0055] The term "target nucleic acid" denotes a nucleic acid of
interest. In some embodiments, the target nucleic acid is one that
a user wishes to determine the presence or absence of. In some
embodiments, the target nucleic acid is one that the user may wish
to specifically attach a cross-linker to. Exemplary target nucleic
acids include DNA, RNA, mRNA, and miRNA.
[0056] The term "cross-linking probe" denotes a molecule that is
capable of selectively binding to a target nucleic acid and then
cross-linking itself to that nucleic acid. While cross-linking
probes can be used to "probe" for specific target nucleic acids in
a sample, a "cross-linking probe" is not limited to this use and
need not include a detectable marker or other detectable aspect.
For example, a cross-linking probe can include only a nucleic acid
probe region and a cross-linker. Such a probe can be used to
obstruct or inactivate a target nucleic acid, rather than for the
detection of the presence or absence of the nucleic acid. Of
course, the probes can include the other aspects disclosed herein
as well.
Cross-Linking Probes
[0057] The upper-left corner of FIG. 1A depicts an embodiment of a
cross-linking probe. This cross-linking probe 201 comprises a
single-stranded initiator region 10 that is linked to a probe
region 20, which includes at least one cross-linker 1. Initially,
cross-linker 1 and/or probe region 20 are/is effectively obstructed
from freely interacting with the environment (in particular, any
part of the sample that is not the intended target sequence) by a
blocking region 25 that is effectively complementary to the probe
region 20 (complementary enough to allow this beneficial blocking
to occur). In the depicted embodiments, there is an optional loop
region 30, which links the probe region 20 to the blocking region
25. As shown in FIG. 1A, combining a target nucleic acid 5 with the
cross-linking probe 201 allows for the initiator region 10 to
hybridize to the target sequence (depicted as process 111), which
allows for branch migration up the cross-linking probe (depicted as
process 112) and thus for the separation of the probe region 20
from the blocking region 25. This then allows for the base-pairing
of the initiator region 10 and the probe region 20 to the target
nucleic acid 5 (depicted as process 11). Following this, in some
embodiments, one can then activate the cross-linker(s) 1 via
photo-activation or from the conformational change, to allow the
cross-linker(s) 1 to form cross-link(s) 2 between the probe region
20 and the target nucleic acid 5 (in particular, the section of the
target that is complementary to the probe region 7). In some
embodiments, the initiator region is immediately adjacent to the
probe region so as to promote effective strand displacement of the
blocking region 25 upon binding of the initiator region 10 to
target.
[0058] As noted above, a specific and tight association between a
target and another molecule can be beneficial in any technique
where a relatively high degree of specificity and tight association
is beneficial. FIG. 1B provides a flow chart outlining a variety of
possible steps in various methods of using such a cross-linking
probe. As shown in FIG. 1B, one can start by providing (e.g.,
making and/or obtaining) a target nucleic acid 300 (or a sample
that is suspected of having a target nucleic acid), providing the
cross-linking probe 310, and combining the two 320. One then allows
(and/or promotes) the selective hybridization of the initiator
region to at least a part of the target nucleic acid 330 and allows
(and/or promotes) the selective hybridization of the probe region
to a second part of the target nucleic acid sequence 340. In
embodiments that employ an activatable cross-linker, one can then
activate the cross-linker 350. One then allows the cross-linker to
cross-link to the target 360. In some embodiments, this can achieve
the result of modifying the functionality of the target nucleic
acid 370. In some embodiments, e.g., when the probe is used for in
situ type methods, it can be useful to have detectable markers
associated with the probe region. In some embodiments, the
detectable marker (and/or the detectable marker region) is already
part of the cross-linking probe and can be linked, directly or
indirectly to the probe region 390. In some embodiments, the
detectable marker region is associated with the probe region via an
amplifier molecule, in process 380. This can be achieved via a
pairing region and a sequence that is complementary to the pairing
region. In some embodiments, a hairpin structure is used as the
amplifier molecule 400. In some embodiments, the pairing region and
pairing region complement are paired via the use of orthogonal
bases that do not hybridize with natural nucleic acids. 410. Once
one or more detectable markers are associated with the probe
region, the sample can be washed to remove any non-cross-linked
probes. In some embodiments, given that the desired probe is
cross-linked to the desired target, ultrastringent wash conditions
can be used 420. Following the wash, one can examine the sample for
the presence or absence of the detectable marker to determine
whether or not the target nucleic acid is present in the
sample.
[0059] While the above sections describe some various general
embodiments, the following section provides more detailed specific
embodiments of using the cross-linking probes.
Additional Cross-Linking Probe Embodiments
[0060] FIG. 2 depicts an embodiment of a cross-linking probe and
its method of use. Initially, one binds the target nucleic acid
with a cross-linking probe 3 (in process 11). The probe molecule 3
is introduced to the sample containing the target nucleic acid. The
exposed initiator region 10 base-pairs to the target 5 at a first
region 6, allowing rapid nucleation with the target (a kinetic
effect) and providing affinity for the target via the formation of
new base-pairs (a thermodynamic effect). After the probe 3
nucleates with the target mRNA via base-pairing at a first region
6, the target 5 base-pairs to the probe region 20 via a branch
migration that opens a hairpin loop (which is an optional
structure). The opening of the hairpin loop provides an entropic
benefit that increases the strength of the interaction between the
probe 3 and the target 5 (as would the dissociation of the blocking
region if there is no loop). In some embodiments, the bases within
the probe region carry activatable cross-linkers 1, which are now
base-paired to the target 5. The blocking region 25 can provide a
stringent specificity check that helps ensure that the bases
carrying activatable cross-linkers 1 primarily (and, in some
embodiments, only) pair to endogenous nucleic acids if they are
specifically paired to the target 5. While not intending to be
limited to theory, it is understood that this is because it is
energetically prohibitive to open the stem to expose the
cross-linkable bases 1 except via a branch migration process in
which the intra-stem base pairs are replaced one-by-one by
intermolecular base-pairs between the probe 3 and the target 5. The
presence of each additional base pair in the stem between the
initiator region 10 and the cross-linkable base(s) 1 increases the
specificity stringency.
[0061] Next, one can covalently cross-link the probe region to the
target in process 12. In some embodiments, one allows sufficient
time for the probe molecules to diffuse into the sample and bind to
target mRNAs. Following this, the covalent cross-linkers 1 are
photo-activated leading to probe-target covalent cross-linking,
which includes at least one cross-link 2. In some embodiments, only
those probes base-paired specifically to target nucleic acids are
covalently linked to the sample. In some embodiments, all other
probes become fused in the closed state due to covalent linking of
the protection and propagation regions. As discussed herein, there
are a variety of mechanisms for activating the cross-linking
process, if one is employing an activatable cross-linker.
[0062] Once the specifically bound probes are covalently linked to
the sample, it is possible to employ one or more ultra-stringent
washes (process 13) that remove a significant amount of all other
probes from the sample to yield exquisite specificity. In some
embodiments, more of the other probes can be removed than would be
typical from traditional washes. In some embodiments, all (or
substantially all) of the non-cross-linked cross-linkable probes
are removed. This can include any probes that are not hybridized to
the sample, that are partially hybridized to the sample, or that
are hybridized but not cross-linked to the sample. In some
embodiments, all probes that are not crossed linked to the target
nucleic acid are removed. In some embodiments, substantially all of
the uncrosslinked probe is removed. In some embodiments,
substantially all of the detectable probe that has not been
cross-linked to a non-probe nucleic acid is removed. In some
embodiments, substantially all of the detectable probe that has not
been cross-linked to a non-probe nucleic acid is removed. In some
embodiments, the only unbound probe that remains after the wash is
readily identifiable as being background. Once the specifically
bound probes are cross-linked to their targets, it is possible to
employ one or more ultra-stringent washes (process 13) to remove
non-cross-linked probes. In some embodiments, substantially all
non-cross-linked probes are removed from the sample. In some
embodiments, 90,% or more of the non-crosslinked probes are
removed, for example 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9%,
99.99% or more of the uncrosslinked probe is removed. In some
embodiments, the background signal per cell is less than the
strength of the amplified signal for a correct target.
[0063] Once a significant amount of the non-crosslinked probe has
been washed away, the sample can be imaged 14. In some embodiments,
fluorescence microscopy can be employed to image the location of
the cross-linked cross-linking probes, thus revealing the location
of the target nucleic acids.
[0064] In some embodiments, the cross-linking probe, instead of
carrying fluorophores on a single-stranded DNA tail, can employ
branched DNA structures or other non-linear DNA structures as
fluorophore carriers. In some embodiments, the detectable marker
region 40 in the cross-linking probe 3 has an independent sequence
from the probe region. Thus, the modular approach shown in FIG. 3
can be used for synthesizing some embodiments of the cross-linking
probe. The detectable marker region 40 and the other part of the
cross-linking probe (25, 30, 20, 10, and 1) are synthesized
separately, and then ligated using nucleic acid 35 to link the two
parts together to produce a complete cross-linking probe. In some
embodiments, the detectable marker region 40 can have an
independent sequence from the target, and thus, the detectable
marker region can be reused for different targets.
[0065] FIG. 4 depicts another embodiment of using a cross-linking
probe. In this embodiment, while the probe 3, lacks a detectable
marker region, the use of an amplifier molecule 4, allows for the
addition of the detectable marker and/or region to be associated
with the target, via the pairing region 140. In this embodiment,
one allows the amplifier molecule 4 to bind to pairing region 140
via a complementary pairing region 142 and/or 144. In this
embodiment, at least a part of the complementary pairing region 144
is blocked by a second blocking region 125. The amplifier molecule
thus undergoes a conformational change, similar to that described
for FIG. 2. One can then cross-link the amplifier molecule 4 to the
cross-linked probe 3 via photo-activation of the cross-linker 22.
Then one can perform an ultra-stringent wash to remove all other
amplifiers from the sample. Thus, in some embodiments, multiple
washes and multiple cross-linker activation steps can be performed.
In some embodiments, multiple amplifier molecules can be used
simultaneously such that each amplifier molecules targets the
single-stranded tail of another species, with intermolecular
covalent linkages formed only in the case of specific interactions.
In some embodiments, those amplification polymers that are
covalently linked to the target nucleic acid would be retained
during the ultra-stringent wash.
[0066] FIG. 5 depicts an embodiment which employs orthogonal base
pairing (between pairing region 150 and a complementary pairing
region 151) to replace the role of conformation change during
amplification in the example above. In some embodiments, the
pairing region 150 has no natural bases appear consecutively in the
pairing region of the amplifier strand 151 (for example natural
bases could alternate with orthogonal bases such as iso-C and
iso-G). In some embodiments, any orthogonal base combination that
will avoid, prevent, or reduce hybridization of the pairing region
to native or natural nucleic acids can be used. In some
embodiments, the orthogonal bases can base-pair to each other (e.g.
iso-C pairs strongly with iso-G) but cannot base-pair to the
natural bases (see, e.g., Collins, M. L., et al., A branched DNA
signal amplification assay for quantification of nucleic acid
targets below 100 molecules/ml. Nucleic Acids Research, 1997.
25(15): p. 2979-2984). As a result, the amplifier molecules cannot
significantly base-pair to endogenous nucleic acids because the
natural bases in the amplifier strand would be able to form only
isolated base-pairs separated by energetically unfavorable interior
loops (i.e. few energetically stacked base pairs could result from
these non-specific interactions). By placing orthogonal bases in
the pairing region 150 (and/or complement 151), one can ensure that
the complementary pairing region 151 (which will contain a sequence
that can hybridize to the orthogonal bases and/or contain the
orthogonal bases) will base-pair primarily (and in some embodiments
only) to the molecule that includes the probe region. The same
approach can be used for base-pairing of multiple amplifier
molecules to form a linear or branched amplification polymer in
which the complementary pairing region contains sufficient
orthogonal nucleotides so as to prevent any substantial or
effective base pairing between the complementary pairing region and
other nucleotides in the sequences. In some embodiments, the
complementary pairing region 151 contains no consecutive natural
bases and intermolecular base-pairing between the amplification
molecules is via a combination of natural and orthogonal base
pairs.
[0067] Once one has an amplifier molecule with an orthogonal
complementary pairing region 151 one allows the amplifier molecule
4 to bind to pairing region 150 via a combination of natural and
orthogonal base pairs. One can then cross-link the amplifier
molecule 4 to the molecule containing, linked to, or associated
with the probe region 20. If the cross-linker 1, requires
activation, then the activation step can also be performed.
Following this, one can then use an ultra-stringent wash to remove
effectively all other amplifier molecules 4 from the sample.
[0068] FIG. 6 depicts another embodiment in which one can employ a
cross-linking probe. As shown in FIG. 6, cross-linkers can be used
in other blocked probe arrangements, such as in hybridization chain
reactions, as discussed in U.S. Pat. Pub. Nos. 20060228733,
20050260635, and 20060234261, the entireties of each of which,
including the discussion of hairpins and their use in hybridization
chain reactions, and HCR itself are incorporated herein by
reference. As can be seen in FIG. 6, in one embodiment, the pairing
region 540 (with subregions 601 and 602) of a probe (only the
pairing region is depicted in FIG. 6, the rest of the probe (which
can be a shielded cross-linking probe) can be on either side of the
pairing region) can be combined with two hairpins (504 and 505)
that each include a crosslinker (597 and 598). The first hairpin
504 includes an initiator region 542 (complementary to 602), a
complementary pairing region 544 (complementary to 601), a loop
region 530, and a blocking region 554 that is complementary to 544.
The second hairpin 505 includes a second initiator region 507 that
is complementary to the loop region 530, another complementary
pairing region 545 that is complementary to 554, a loop region 531,
which is complementary to the initiator region 542, and a blocking
region 555, that is complementary to region 545 and can be the same
as region 554. As shown in the figure, these monomers can be
allowed to form a polymer which will include the crosslinkers (598
and 597). In turn, the polymer can then be crosslinked, resulting
in a crosslinked polymer. In some embodiments, the HCR scheme will
also work when some or all the polarities of the strands are
reversed (arrow at the opposite ends of the probe hairpin and the
amplification hairpins). The stars in FIG. 6 represent optional
detectable markers.
[0069] In some embodiments, any of the described embodiments herein
will also work when some or all of the polarities are reversed.
[0070] In some embodiments one can employ a shielded cross-linking
probe in situations where the size of the fluorophore-carrying
segment hinders penetration into the sample. In such embodiments,
nucleated dendrimers (as described in P. Yin, H. M. T. Choi, C. R.
Calvert, N. A. Pierce. Programming biomolecular self-assembly
pathways. Nature, 451:318-322, 2008; U.S. Pat. Pub. Nos.
20090011956; and 20060234261, the entireties of each of which,
including the various teachings regarding dendritic growth and HCR,
are incorporated herein by reference) can be used to deliver one or
more fluorophores bound to the binding probe (or a molecule
associated therewith). In some embodiments, orthogonal isoC/isoG
bases can be interspersed with natural bases to ensure that the
dendrimer components do not base-pair non-specifically with native
nucleic acids. After (or with) the assembly of the amplification
dendrimer attached to the target nucleic acid/binding probe
complex, the dendrimer can be covalently cross-linked to the
binding probe (or molecules associated or linked thereto), and each
branch of the dendrimer can be covalently linked to its parent
branch. In such embodiments, the initial components of the branches
can all (or some fraction thereof) include a cross-linker which can
(but need not be) activatable. In some embodiments, a subsequent
stringent wash can be applied to remove all other
non-specifically-bound amplification molecules. In some
embodiments, cross-linkers can be used in other blocked probe
arrangements, such as in hairpins in hybridization chain reactions,
as discussed in U.S. Pat. Pub. Nos. 20060228733 and 20060234261,
the entireties of both of which, including the discussion of
hairpins and their use in hybridization chain reactions, are
incorporated herein by reference.
Alternative Embodiments of Components
[0071] While, given the present disclosure, one of skill in the art
will appreciate that there are a variety of alternative embodiments
for each of the herein noted components, the following section
briefly outlines some explicit alternative embodiments for some of
these components.
[0072] As noted above, orthogonal nucleotides can be used for a
variety of purposes in a cross-linking probe or the molecules that
are associated therewith. In some embodiments, these orthogonal
nucleotides can be used to improve or ensure specificity between an
amplifier molecule and a molecule that includes a probe region. In
some embodiments, they can be used to allow specificity for the
parts of a dendrimer. In some embodiments, they can be used to
provide for a blocking region that can block or obstruct a
cross-linker, while preventing the cross-linker from forming a
cross-link with the blocking region. In such an embodiment, any
nucleotide that will not effectively cross-link with the
cross-linker can be used. Indeed, in some embodiments, a molecule
that only contains the backbone of the nucleotide and/or lacks one
or more atoms involved in cross-linking (such as the bases) can be
used.
[0073] The initiator region allows for the initial priming of the
cross-linking probe to the target nucleic acid and can allow for
subsequent strand displacement of the blocking region. This is not
required for all embodiments. In some embodiments the initiator
region is at least 2 and typically not more than 200, but can be as
many as 1000 nucleotides or more. In some embodiments, the
initiator is immediately adjacent to the probe region. In some
embodiments there can be an intervening structure, as long as
effectively specific strand displacement is maintained.
[0074] The probe region ensures that the molecule that the
cross-linker cross-links to is the specific nucleic acid that is
desired. The probe region can be a single continuous nucleic acid
or it can be broken into two or more parts. In some embodiments,
the probe region will include 3-50 nucleotides. In some
embodiments, the probe region is less than 300 nucleotides. In some
embodiments, the probe region is less than 200 nucleotides. In some
embodiments, the probe region is less than 100 nucleotides. In some
embodiments, the probe region is extended into the loop to further
lock the probe in the open conformation once it is bound
specifically to the target mRNA.
[0075] As noted above, not all cross-linking probes require
activatable cross-linkers, loop regions, and/or initiator regions.
In some embodiments, the cross-linking probe includes a probe
region, a cross-linker, and a blocking region, that reduces the
accessibility of the cross-linker to external nucleic acids. In
some embodiments, the cross-linking probe further includes an
initiator region. In some embodiments the initiator region is
immediately adjacent and linked to the probe region such that
binding of a target to the initiator region allows for strand
displacement against a molecule hybridized to the probe region
(e.g., the blocking region). In some embodiments, the cross-linking
probe further comprises a loop region that links the probe region
and the blocking region. In some embodiments, the cross-linking
probe further comprises a detectable marker. In some embodiments,
the cross-linking probe comprises a detectable marker region. In
some embodiments, the cross-linking probe comprises a pairing
region. The pairing region can be linked to the blocking region or
can be linked to the probe region or loop region. In some
embodiments, the pairing region can be or be part of the looped
region (and thus will be accessible for hybridization upon
displacement of the blocking region and the probe region.
[0076] In some embodiments, the blocking region is the same length
as the probe region. In some embodiments, the blocking region is
shorter than the probe region, but long enough to effectively
reduce or inhibit non-target based cross-linking. In some
embodiments, the blocking region comprises one or more nucleic acid
strands and thus, comprises multiple subparts. In some embodiments,
the blocking region has orthogonal bases. In some embodiments a
part of the blocking region (e.g., the portion that might otherwise
be at risk of cross-linking with the cross-linker) lacks those
atoms or residues that allow for cross-linking. For example, in
some embodiments, the blocking region only includes a backbone at
those sections and lacks a base for cross-linking to.
[0077] As will be appreciated by one of skill in the art, the
cross-linking probe and its use can be employed for any target
nucleic acid, such as those nucleic acids that one may wish to
observe (such as in in situ hybridization) or those that one may
target for covalent modification (which would naturally impair the
functionality of that nucleic acid). Exemplary target nucleic acids
include, for example: RNA, DNA, mRNA, and miRNA. The target nucleic
acids can be from any organism, including, but not limited to,
mammals, mice, rats, primates, humans, etc. In some embodiments,
the target nucleic acid remains in the host cell or subject. Thus,
in some embodiments, the method can be performed in vivo, ex vivo,
or in vitro. In some embodiments, the method is performed in a
cell. In some embodiments, the method is performed in tissue. In
some embodiments, the tissue is in a living host.
[0078] In some embodiments, the amount of the cross-linking probe
provided is sufficient to allow for the determination of the
presence or absence of a target nucleic acid. In some embodiments,
the amount of the cross-linking probe provided is sufficient to
allow for the determination of the amount of a target nucleic acid.
In some embodiments, the amount of the cross-linking probe provided
is sufficient to allow for binding and cross-linking to at least 1%
of the target nucleic acid present in the sample to be exposed to
the cross-linking probe, for example 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,
99, 99.9, or greater percent of the nucleic acid target is
cross-linked to a cross-linking probe, including any amount greater
than any of the preceding values and any range defined between any
two of the preceding values. In some applications, the
concentration of probe is higher than the concentration of the
target (e.g., imaging rare mRNAs). In some applications, the probe
concentration may be lower (e.g., detecting DNA or RNA on a
chip)
[0079] In some embodiments, one or more cross-linkers can be used
in a cross-linking probe. In some embodiments, the cross-linkers
are activatable. In some embodiments, the activatable cross-linker
is a photo-activatable cross-linker. Exemplary photo-activatable
cross-linkers include psoralens (Pieles, U. and Englisch, U.
Psoralen covalently linked to oligodeoxyribonucleotides: synthesis,
sequence specific recognition of DNA and photo-cross-linking to
purimidine residues of DNA. Nucleic Acids Research, 1989. 17: p.
285-299), thiols (Killops, K. L., Campos, L. M., Hawker, C. J.
Robust, Efficient, and Orthogonal Synthesis of Dendrimers via
Thiol-ene "Click" Chemistry. Journal of the American Chemical
Society, 2008. 130: p. 5062-5064), and halogenated nucleobases
(Willis, M. C., et al. Photocross-linking of
5-Iodouracil-Substituted RNA and DNA to Proteins. Science, 1993.
262: p. 1255-1257) (all of which are incorporated herein by
reference in their entireties and especially in regard to
psoralens, thiols, and halogenated nucleobases). In some
embodiments, any compound that forms interstrand cross-links when
activated by ultraviolet light can be a photo-activatable
cross-linker. In some embodiments, the photo-activatable
cross-linker only cross-links when exposed to ultraviolet light,
reducing the risk of premature cross-linking that could reduce
probe sensitivity.
[0080] In some embodiments, multiple cross-linkers can be used in a
single probe such that the overall yield of probes that form at
least one covalent bond to the target is close to unity. In some
embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or more cross-linkers are
used. The cross-linker(s) can be placed on either end or within the
probe region. The placement of the cross-linkers within the probe
region is especially advantageous. In some embodiments, where the
probe region is comprised of two or more subparts, the cross-linker
can be at a terminal end of one or more of the subparts.
[0081] In some embodiments, the cross-linking and cross-linker are
non-promiscuous in that the cross-link is formed only between the
probe region and its target and not simply to any molecule that is
in the proximity of the bases that carry cross-linkers. In some
embodiments, the efficiency is such that at least 80, 90, 95, 99,
99.5, 99.9, 99.99% or more of the cross-linked probe is
cross-linked to a sequence that hybridizes to the probe region upon
activation of the cross-linker (if activation is required). For
example, in some embodiments 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, or 100%
of the cross-linking probe is cross-linked to a target nucleic acid
that will hybridize to the probe region, including any range
greater than any of the preceding values and any range defined
between any two of the preceding values. In some in vitro
applications, there need be no lower limit for the above noted
percentages.
[0082] In some embodiments, the cross-linker can be activated by
the conformation change of the molecule. For example, in the probe
region, bases carrying the cross-linker form Watson-Crick pairs
between the probe region and the blocking region that are then
replaced with wobble pairs to the mRNA target upon the binding of
the probe region to the target. The cross-linkers have the property
that they covalently link to their wobble-complement but not to
their Watson-Crick complement (e.g., a G base carrying a
cross-linker covalently binds to a U base but not to a C base). An
example of such a cross-linker has been provided in (Coleman, R. S.
and Pires, R. M. Covalent cross-linking of duplex DNA using
4-thio-2'-deoxyuridine as a readily modifiable platform for
introduction of reactive functionality into oligonucleotides.
Nucleic Acids Research, 1997. 25: p. 4771-4777; the entirety of
which is incorporated herein by reference, including the disclosure
regarding the cross-linker). In some embodiments, the cross-linker
need not be activatable. Rather, the cross-linker can be one that
would otherwise readily cross-link, if not for the presence of the
blocking region. In such embodiments, one or more sections or
subparts of the blocking region are selected so as to be inert to
cross-linking (and thus avoiding the formation of initially
cross-linked cross-linking probes) and the removal of the blocking
region allows for the cross-linking of the cross-linker to the
nucleic acid that displaces the blocking region. In some
embodiments, these sections are made of orthogonal nucleotides or
can simply be linkers or involve a backbone without the bases to
which the cross-linkers cross-link to.
[0083] In some embodiments, the probe region and the blocking
region are connected by a loop region. The loop region can include
one or more nonnatural nucleotides or orthogonal nucleotides. In
some embodiments, the loop region is a linker or polymer and need
not be nucleotide based, as long as it can link the probe region
and the blocking region. In some embodiments, the loop includes
nucleotides that can bind to the target nucleic acid, thereby
further enhancing the hybridization.
[0084] In some embodiments, the cross-linking probe is washed away
from the sample. In some embodiments, the washing conditions are
more stringent than are used for situations in which a probe is
simply hybridized to a target. Thus, the washing conditions can be
higher than allowed for typical in situ washes.
[0085] In some embodiments, the presence or absence of the
detectable marker can be detected by imaging a sample. In some
embodiments, an actual image of the sample is not required and the
sample can simply be reviewed for the presence or absence of any
detectable marker.
[0086] In some embodiments, the detectable marker is selected from
one or more of the following: fluorescent markers (including
organic fluorophores, fluorescent nucleoside analogs, and inorganic
semiconductor nanocrystals), chromogenic chemical substrates or
enzymes, metallic particles, organic dyes, haptens for
immunochemistry, commercially available fluorophores, including
quantum dots. Of course, in some embodiments, the detectable marker
need not emit radiation, for example, when surface plasmon
resonance is used to detect binding, the detectable marker can be
the probe region itself.
[0087] In some embodiments, one or more of the herein disclosed
embodiments are employed on a "DNA chip"-type application. For
example, in some embodiments the probe region (which is shielded as
described herein) is attached to the chip and the target is
crosslinked to the chip, via the probe region only if it displaces
the blocking region (which could then be washed away from the
chip). In some embodiments, the probe region can be immobilized on
any solid surface and then employed to pull out or detect the
target nucleic acid.
[0088] Existing in situ hybridization bioimaging methods share the
weakness that the background signal is raised by amplification of
probes that bind non-specifically within the sample (Qian, X., L.
Jin, and R. V. Lloyd, In situ hybridization: basic approaches and
recent development. The Journal of Histotechnology, 2004. 27(1): p.
53-67). In some embodiments, the shielded cross-linking methods or
probes can be employed to avoid or reduce this issue. One of skill
in the art will also appreciate that in other embodiments, the
cross-linking probes or methods do not avoid or reduce this
issue.
[0089] In some embodiments, the use of the present cross-linking
probes allows the detailed analysis of genetic regulatory
processes.
[0090] In some embodiments, the probe and/or technique is specific,
in that fluorescence signals are generated at the site of a target
molecule (and in some embodiments can minimize or reduce false
positives).
[0091] In some embodiments, the probe and/or technique is
sensitive, in that the fluorescence signal is strong enough to
enable imaging of single target molecules (and in some embodiments
can minimize false negatives).
[0092] In some embodiments, the probe and/or technique is
multiplexed, in that the amplification of all probe species can be
performed simultaneously in parallel (and in some embodiments can
enhance efficiency and reduce sample degradation).
[0093] In some embodiments, the probe and/or technique is
quantitative, in that it allows quantized fluorescent signaling per
target molecule (and in some embodiments can allow for relative
quantification of target abundance within samples).
[0094] In some embodiments, regardless of the nature of any
subsequent downstream amplification step, the present probe concept
is superior to other existing probe strategies with regard to
conferring specificity.
[0095] In embodiments in which the probes carry sufficiently many
fluorophores of sufficient brightness and the detection device is
sufficiently sensitive, then no further amplification is necessary.
In some embodiments, additional amplification is desirable to
increase the signal strength associated with each target molecule.
In performing this amplification, it is valuable to retain the
specificity that was achieved during the detection stage.
[0096] In some embodiments, the above detection and amplification
schemes can be used to target multiple nucleic acid targets
simultaneously by using probe and amplifier sequences that are
orthogonal in sequence space. Thus, multiplexing becomes
possible.
[0097] In some embodiments, the above detection and amplification
schemes can be used to produce quantized fluorescent signal
strength associated with each target nucleic acid that has been
covalently linked to a probe molecule (and optionally to a number
of additional fluorescently-labeled amplifier molecules).
[0098] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
EXAMPLES
Example 1
mRNA Cross-Linking
[0099] This example illustrates the use of a cross-linking probe to
attach a cross-linker to target mRNA.
[0100] One first obtains a cross-linking probe that includes an
initiator region, a probe region that hybridizes to the desired
target mRNA, a blocking region that obstructs the cross-linker when
bound to the probe region, and a light activatable cross-linker.
One then adds the cross-linking probe to the cell or tissue that is
believed to include the target mRNA and allow the cross-linking
probe to hybridize to the target mRNA. One then activates the
cross-linker by irradiating the cell or tissue. Following this, the
cross-linker will cross-link to the target mRNA, resulting in a
cross-linked mRNA target.
Example 2
In Situ Hybridization
[0101] This example illustrates a use of a cross-linker probe in an
in situ hybridization. One performs the steps outlined in Example
1, except that the cross-linking probe also initially includes a
detectable marker region (with one or more detectable markers) that
is attached to the blocking region.
[0102] Following irradiation, one then washes the tissue or cell
using an ultrastringent wash containing denaturing chemicals
including formamide and performing the wash at elevated
temperatures. Following this, one then images the remaining
detectable markers in the cell or tissue in order to identify
whether or not, where, and how much of the target nucleic acid is
present.
Example 3
mRNA In Situ Hybridization Using a Hairpin Amplifier Molecule
[0103] This example illustrates a use of a cross-linker probe in an
in situ hybridization. One performs the steps outlined in Example
1, except that the cross-linking probe also initially includes a
pairing region that is attached to the blocking region.
[0104] Following the irradiation, one then washes the tissue or
cell via an ultrastringent wash protocol to remove a significant
portion of the unbound cross-linking probe. Following this, one
then adds the amplifier molecule, which includes a detectable
marker region (with decteable markers) and a complementary pairing
region that will bind to the pairing region on the cross-linking
probe. The amplifer molecule can have the general structure
depicted in FIG. 4, and the amplifier molecule can include at least
one photoactivatable cross-linker in the complementary pairing
region. Following this, one can then irradiate the cell or tissue a
second time to cross-link the cross-linker in the amplifier
molecule. Following the second irradiation, one then washes the
tissue or cell to remove a significant portion of the unbound
amplifier molecule.
[0105] Following this, one then images the remaining detectable
markers in the cell or tissue in order to identify whether or not,
where, and how much of the target nucleic acid is present.
Example 4
mRNA Hybridization Using a Orthogonal Base Containing Amplifier
Molecule
[0106] This example illustrates a use of a cross-linker probe in an
in situ hybridization. One performs the steps outlined in Example
1, except that the cross-linking probe also initially includes a
pairing region that is attached to the blocking region.
[0107] Following the irradiation, one then washes the tissue or
cell via an ultrastringent wash protocol to remove a significant
portion of the unbound cross-linking probe. Following this, one
then adds the amplifier molecule, which includes a detectable
marker region (with detectable markers) and a complementary pairing
region that will bind to the pairing region on the cross-linking
probe and that has no consecutive natural nucleotides. The amplifer
molecule can be that depicted in FIG. 5, and the amplifier molecule
will include a complementary pairing region that can bind to the
pairing region in the cross-linking probe. Following this, one then
washes the tissue or cell to remove a significant portion of the
unbound amplifier molecule.
[0108] Following this, one then images the remaining detectable
markers in the cell or tissue in order to identify whether or not,
where, and how much of the target nucleic acid is present.
Example 5
[0109] Tethered psoralens were prepared via two
commercially-available products: a trimethylpsoralen (TMP)
phosphoramidite 1007 (see, e.g., FIG. 7) for incorporation at the
5'-terminus of an oligonucleotide during solid-phase synthesis and
a succinimidyl ester derivative 1008 (see FIG. 7) of psoralen that
can be conjugated to an amine-modified oligonucleotide
post-synthetically.
[0110] 18 mer probes were either: (a) synthesized using 5'-terminal
trimethylpsoralen phosphoramidite (Glen Research), or (b) labeled
post-synthetically by conjugation of succinimidyl ester of 8-yloxy
psoralen 8 (SPB, Pierce) to amine modified thymine. Probes were
hybridized to 21 mer targets and irradiated with 365 m UV light for
30 min (30 mW/cm2, TMP saturated after 5 minutes, SPB 25 minutes),
then analyzed by denaturing gel electrophoresis. The gels were
post-stained with SyBr Gold (Invitrogen) and crosslinking yield
determined by comparing the intensity of non-crosslinked target
band (red box) to non-irradiated controls. The results are shown in
FIG. 7.
[0111] The efficacy of both molecules in cross-linking DNA and RNA
targets in vitro were examined. When attached to a probe by a
2-carbon linker, TMP cross-linked duplex DNA with moderate
efficiency, but was much less effective at cross-linking to an RNA
target. The results for both of these molecules are shown in FIG.
7. The 6-carbon linker enabled TMP to efficiently bind both DNA and
RNA targets. The succinimidyl derivative of psoralen can be added
to an oligonucleotide at any position, but the cross-linking yields
were unacceptably low for DNA and lower still for RNA.
[0112] While the present teachings have been described in terms of
these exemplary embodiments, the skilled artisan will readily
understand that numerous variations and modifications of these
exemplary embodiments are possible without undue experimentation.
All such variations and modifications are within the scope of the
current teachings.
[0113] Although the disclosed teachings have been described with
reference to various applications, methods, kits, and compositions,
it will be appreciated that various changes and modifications can
be made without departing from the teachings herein and the claimed
invention below. The foregoing examples are provided to better
illustrate the disclosed teachings and are not intended to limit
the scope of the teachings presented herein.
[0114] Unless otherwise indicated, the singular use of various
words, including the term "an" or "an" denotes both the option of a
single or more than one. In addition, the use of the term "and/or"
denotes various embodiments that include: both options, either
option in the alternative, or the combination of either option in
the alternative and both options. When describing various
combinations, kits, probes, methods, etc., it will be understood
that unless otherwise stated, the combinations are described as
comprising, consisting of, and consisting essentially of. This does
not apply to the claims or to situations in the specification where
the term "consisting of" is used.
Incorporation by Reference
[0115] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application, including but not
limited to defined terms, term usage, described techniques, or the
like, this application controls.
Equivalents
[0116] The foregoing description and Examples detail certain
specific embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
2121DNAArtificial SequencePrimer 1actaaccgat agcggctacc g
21218DNAArtificial SequencePrimer 2tagccgctat cggttagt 18
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